Summary Toxoplasma gondii is an obligate intracellular apicomplexan parasite causing severe opportunistic infections. Current drugs are prone to induce hypersensitivity, especially upon long-term use. Under this proposal the unique cell division process will be interrogated to identify putative new drug targets. Toxoplasma divides by a distinct internal budding process whereby two daughter parasites are assembled within a mother cell. The cortical membrane skeleton composed of flattened alveolar vesicles supported by an epiplastin protein network and 22 subpellicular microtubules (MTs) is nucleated on the centrosomes and assembles in an apical to basal direction. In the second half of division the posterior end of the daughter buds (i.e. the basal complex or BC) starts to taper driven by Myosin J (MyoJ). Absence of MyoJ only modestly impact parasite viability, even while it leaves the BC somewhat unconstricted, fitting classic data on cell division resistance to actin depolymerizing agents. However, preventing assembly of the BC altogether by depleting or overexpressing the BC scaffolding protein MORN1 results in parasites with fraying MTs unable to complete cell division and has dramatic impact on viability. To unravel this intriguing process, under an R21 grant the BC was proteomically dissected through proximity dependent biotinylation (BioID) on 8 BC components. This revealed 4-5 different protein complexes aligning with the ultrastructure. Two key observations are further pursued under this proposal: 1. A putative MT Associated Protein, MAP1B-L1, appears to assemble on the (+)-ends of the subpellicular MTs and is essential for BC assembly and parasite viability; 2. Several kinases and phosphatases identified indicate the BC is regulated by differential phosphorylation. Under Aim 1 MAP1B-L1 and another critical BC MAP dubbed MAP1B- L2 will be tested for MT binding capacity by generating deletion mutants in the parasite, in vitro using the identified MT binding domains, and by exogenous expression in the Xenopus leavis axon guidance model as relevant to related MAPs. Under Aim 2 we will pursue four additional candidates identified in the BioID approach with a likely essential function, which are all hypothetical proteins narrowly conserved in internally budding parasites and harbor putative adhesion domains. In addition, we will apply fast acting TurboID on BC components transiently associating with the assembling BC like MAP1B-L1 as these were likely undersampled in the current dataset, yet define the essential step of the BC in cell division. Under Aim 3 we will subject 2 kinases and 1 phosphatase to synthetic lethality screening using the genome wide CRISRP/Cas9 library. Preliminary data of the first kinase tested already demonstrates experimental feasibility and revealed interesting new insights. Combining the proteomic and genetic data sets is expected to provide a solid basis to assemble the wiring diagram of the BC. In the current working model the BC is first assembled on the MT (+)-ends, followed by recruitment of adhesion proteins to keep the MT-ends together. Overall, this is expected to deliver exciting new insights into internal budding, how it differs from schizogony, and could highlight new drug targets.